Project Summary/Abstract FACT is a complex of two proteins that are found in all eukaryotes, including humans, and is essential for viability in all organisms tested. Nucleosomes are the fundamental subunit of chromatin, which is the DNA packaging in eukaryotes. Their properties govern the flow of information from the genome. The goals of this project are to determine how FACT causes nucleosomes to adopt an alternative structure and to learn when and where the ability to form and resolve such structures is useful in living cells. FACT is unique among factors that regulate chromatin because it does not modify the histones that make up nucleosomes, it does not use an energy source to move the histone cores along the DNA, and it works through a mechanism that has not been observed with any other histone chaperone. FACT has been identified in a wide range of systems biology studies seeking factors that regulate cellular physiology, promote normal embryonic development, and allow stem cells to maintain pluripotency. Both the mechanism of FACT action and the circumstances in which it is used are therefore of high importance for understanding basic eukaryotic physiology. An integrated approach using a yeast model system is proposed for these studies to take advantage of the range of tools available in these organisms. The first aim of the proposal is to analyze the properties of nucleosomes that have been altered by FACT to produce what is called a reorganized form. This structure is significantly different from canonical nucleosomes, but its nature remains poorly characterized. Purified histones and DNA fragments labeled with fluorescent dyes will be used to probe the properties of reorganized nucleosomes, and mutant histone and FACT proteins will be used in these assays to allow the results to be compared with the properties of FACT in living cells. The second aim is to characterize the roles of FACT by identifying mutations that make it easier or harder to tolerate FACT defects using whole-genome sequencing, and by asking how distinct mutations in FACT and other histone chaperones affect the ability to maintain repression of cryptic promoters and promote stability of distinct chromatin states using quantitative PCR measurements of transcripts and chromatin immunoprecipitations (ChIPs). The third aim is to ask how FACT affects chromatin structure near origins of DNA replication, how this contributes to regulating origin firing, and to test a hypothetical role for FACT in depositing new chromatin after the genome is duplicated during replication. This aim uses some established ChIP technology but also requires development of some novel methods.